Toxoplasma gondii is an ubiquitous Apicomplexen intracellular parasite which is responsible for several important clinical syndromes in humans. In the setting of the acquired immunodeficiency syndrome (AIDS) this organism causes encephalitis which is often fatal and has been associated with headache, seizures and personality changes. If acquired during pregnancy infection can result in the syndrome of congenital toxoplasmosis with attendant encephalitis, mental retardation, chorioretinitis and in severe cases death. In both conditions reactivation of the latent encysted stage of the organism (bradyzoite) to the active replication form (tachyzoite) is associated with progression of disease and is directly implicated in the pathology that attends this infection. Despite major advances in our understanding of tachyzoite antigens little is known about bradyzoite specific antigens and the control mechanisms for the transition of bradyzoites to tachyzoites. We have developed an in vitro technique which allows study of this transition. In addition, by using PCR and mutant strains of T. gondii we have made cDNA libraries that contain bradyzoite specific genes and have developed techniques and have produced Mabs that are bradyzoite specific. We will extend this in vitro model to astrocyte cultures that more faithfully recapitulates an encephalitis model. We plan, during the course of this proposal, to study the factors involved in the development of bradyzoites in vitro with a particular emphasis on the effect of nitric oxide on this development. Preliminary experiments suggest that heat shock proteins (hsps) are also an important factor in development. Observations on in vitro development suggest that during early development the matrix and organization of a parasitophorous vacuole containing bradyzoites is different from a vacuole containing tachyzoites. We plan on characterizing several of these bradyzoite specific genes. Once cloned we will make knock out mutations of these genes in T. gondii, these mutations should aid in establishing their functional importance. These studies should extend our knowledge of this important pathogenic stage of this organism and may point to new strategies for the elimination of the bradyzoite stage of the organism.